Instructions for use

Title Studies on the function of ebolavirus glycoprotein-specific antibodies

Author(s) 古山, 若呼

Issue Date 2017-03-23

DOI 10.14943/doctoral.k12612

Doc URL http://hdl.handle.net/2115/65648

Type theses (doctoral)

File Information Wakako_Furuyama.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

Studies on the function of ebolavirus

glycoprotein-specific antibodies

（エボラウイルス表面糖蛋白質に対する

抗体の機能に関する研究）

Wakako FURUYAMA

i

Contents

Abbreviation -------------------------------------------------------------------------------------------- 1

Preface ---------------------------------------------------------------------------------------------------- 4

Chapter I:

Discovery of an antibody for pan-ebolavirus therapy

Introduction -------------------------------------------------------------------------------------------- 7

Materials and Methods ------------------------------------------------------------------------------- 8

Viruses and cells

Purification of virus-like particles (VLPs) for immunization

Generation of MAb 6D6

Neutralization tests

Enzyme-linked immunosorbent assay (ELISA)

Selection of escape mutants and identification of the putative epitope

Purification and fluorescent-labeling of VLPs

Imaging of attachment, internalization and membrane fusion of DiI-labeled VLPs

Passive immunization and protective efficacy in mice

Statistical analysis

Results ------------------------------------------------------------------------------------------------ 15

In vitro properties of MAb 6D6

Identification of the putative 6D6 epitope

Mechanism of the neutralizing activity of 6D6

Protective efficacy of MAb 6D6 in mouse models

Discussion -------------------------------------------------------------------------------------------- 26

ii

Summary ---------------------------------------------------------------------------------------------- 29

Chapter II:

Fcγ-receptor IIa-mediated Src signaling pathway is essential for the antibody-

dependent enhancement of Ebola virus infection

Introduction ------------------------------------------------------------------------------------------- 30

Materials and Methods ------------------------------------------------------------------------------ 32

Viruses and cells

Generation of FcγRIIa- or FcγRIIaΔCT-expressing Jurkat T cells

ADE assays

Purification and DiI-labeling of VLPs

Imaging of attachment and internalization of DiI-labeled VLPs

Dextran uptake assays

Inhibitor treatments

Generation of Src or Syk knockdown K562 cells

Phosphorylation assay

Statistical analysis

Results ------------------------------------------------------------------------------------------------ 40

Cytoplasmic tail of FcγRIIa is required for ADE of EBOV infection

Cytoplasmic tail of FcγRIIa is important for enhanced viral uptake

Intracellular signaling via Src family PTKs contributes to ADE of EBOV infection

VLP-MAb complexes activate phosphorylation of Src in K562 cells

The ADE of EBOV entry depends on phagocytosis and/or macropinocytosis mediated by

Src family PTKs

Discussion ---------------------------------------------------------------------------------------------- 64

iii

Summary ----------------------------------------------------------------------------------------------- 68

Conclusion ----------------------------------------------------------------------------------------------- 69

Acknowledgement ------------------------------------------------------------------------------------- 71

和文要旨-------------------------------------------------------------------------------------------------- 73

References ----------------------------------------------------------------------------------------------- 76

1

Abbreviations

ADE antibody-dependent enhancement

Alexa647-labeled Dx10 Dextran, Alexa Fluor 647, 10,000 MW

BDBV Bundibugyo virus

BSA bovine serum albumin

Btk Bruton’s tyrosine kinase

CT cytoplasmic tail

CTR IgG control IgG

DAPI 4',6-diamidino-2-phenylindole, dihydrochloride

DC-SIGN dendritic cell-specific ICAM-3-grabbing non-integrin

DiI 1,1'-dioctadecyl-3,3,3',3'-

tetramethylindocarbocyanine perchlorate

DMEM Dulbecco’s modified Eagle’s medium

DMSO Dimethyl sulfoxide

ELISA Enzyme-linked immunosorbent assay

EBOV Ebola virus

EVD Ebola virus disease

FCS fetal calf serum

FcγR Fcγ receptor

FFU focus forming units

GFP green fluorescent protein

GP glycoprotein

HEK human embryonic kidney

hMGL human macrophage galactose-type C-type lectin

2

HR1 heptad repeats 1

HR2 heptad repeats 2

IC50 50% inhibitory concentrations

IFL internal fusion loop

ITAM immunoreceptor tyrosine-based activation motif

IU Infectious unit

MARV Marburg virus

MAb monoclonal antibody

MLR mucin-like region

NP nucleoprotein

NPC1 Niemann-Pick C1

OD optical density

PBS phosphate-buffered saline

PI3K phosphatidylinositol 3-kinase

Plat-GP Platinum-GP

PTK protein-tyrosine kinase

Ras rat sarcoma

RBD receptor binding domain

RESTV Reston virus

RPMI Roswell Park Memorial Institute

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel

electrophoresis

Src sarcoma

SUDV Sudan virus

3

Syk spleen tyrosine kinase

TAFV Taï Forest virus

TBS Tris-buffered saline

TBST Tris-buffered saline containing 0.1% Tween 20

TIM-1 T-cell immunoglobulin and mucin domain 1

VLP virus-like particle

TM transmembrane

VSV vesicular stomatitis virus

4

Preface

Ebolaviruses, members of the family Filoviridae, cause severe hemorrhagic

fever in humans and nonhuman primates, with human case fatality rates of up to 90% 1,2.

The latest epidemic of Ebola virus disease (EVD) in West Africa, ebolaviruses pose a

significant public health concern. However, an effective prophylaxis or treatment for

EVD is not yet commercially available. Five distinct species are known in the genus

Ebolavirus; Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, Bundibugyo

ebolavirus, and Reston ebolavirus, represented by Ebola virus (EBOV), Sudan virus

(SUDV), Taï Forest virus (TAFV), Bundibugyo virus (BDBV), and Reston virus

(RESTV), respectively 3. In the last decade, EBOV, SUDV, and BDBV have caused EVD

outbreaks with increased frequency in Central and West Africa 1,2,4.

Ebolaviruses express a single transmembrane glycoprotein (GP) that is

responsible for both receptor binding and membrane fusion. GP undergoes proteolytic

cleavage by host proteases such as furin, resulting in two subunits, GP1 and GP2, which

are linked by a disulfide bond 5,6. The GP1 subunit (amino acids 33-501) contains the core

of the glycoprotein, a receptor binding domain (RBD), a glycan cap, and a large mucin-

like region (MLR) that extends around the RBD 7. The GP2 (amino acids 502-676)

subunit contains the internal fusion loop (IFL), heptad repeats 1 and 2 (HR1 and HR2),

the transmembrane region (TM), and the cytoplasmic tail (CT) 8. EBOV entry is initiated

by viral attachment to cell surface molecules such as T-cell immunoglobulin and mucin

domain 1 (TIM-1) and C-type lectins 9,10, followed by internalization of the virus particle

into cells via micropinocytosis 11-13. In the late endosome, EBOV GP is cleaved by host

proteases such as cathepsins L and B 14,15, and then binds to the receptor, Niemann-Pick

C1 (NPC1), followed by membrane fusion 8,16-19.

5

Since GP is the only surface protein of ebolaviruses and essential for viral entry

into host cells, it is the sole target of neutralizing antibodies. In the past, many GP-specific

monoclonal antibodies (MAbs) including neutralizing antibodies, have been generated 20-

23. Previous studies have shown that neutralizing antibodies against EBOV GP reduce

virus infectivity through blocking receptor binding (i.e., interaction between GP and

NPC1), cathepsin proteolysis, or membrane fusion 24,25. Importantly, passive

immunization with some GP-specific MAbs protects experimental animals, such as

rodents and nonhuman primates, from lethal EBOV infection 26-31. Indeed, a human MAb

cocktail against EBOV GP was used for the treatment of EVD in patients during the latest

outbreak in West Africa 32-35. However, most of the previously tested MAbs are EBOV-

specific and MAb therapy against other members of the genus Ebolavirus (i.e., SUDV

and BDBV) is not yet available.

On the other hand, some GP-specific MAbs reportedly induce antibody-

dependent enhancement (ADE), a phenomenon in which viral infectivity is increased by

virus-specific antibodies. ADE has been observed in vitro for a large number of viruses

36-38 and it has interfered with vaccinations against some viruses. In addition, some human

sera convalescent from EVD contain ADE antibodies 22. ADE of EBOV infection

depends on the cross-linking of virus-antibody complexes to the cellular Fcγ receptor IIa

(FcγRIIa) or complement component C1q and its ligand 22,23,39. These receptors are

known to activate various signaling pathways that lead to the reorganization of the actin

cytoskeleton and membrane remodeling 40,41. However, little is known about the

molecular mechanisms underlying ADE of EBOV infection.

In this study, I describe two MAbs with different functions: neutralization and

ADE. In chapter I, a broadly cross-reactive GP-specific MAb, 6D6, was generated and

6

analyzed for its neutralization mechanism and protective potential. In chapter II, the

contribution of FcγRIIa-mediated signaling to EBOV ADE was investigated, and the

function of the signaling pathway was analyzed.

7

Chapter I:

Discovery of an antibody for pan-ebolavirus therapy

Introduction

Several studies have demonstrated that administration of EBOV GP-specific

antibodies protects nonhuman primates from lethal EBOV infection and may constitute a

leading treatment option for EVD in humans 26-30. During the West African EVD outbreak,

EBOV GP-specific MAb cocktails (e.g., ZMapp) 26 were used to treat several patients 32-

35. However, most of characterized therapeutic MAbs to date are EBOV GP-specific and

cross-neutralizing activity against any other ebolavirus species (e.g., SUDV and BDBV)

has not been demonstrated due to antigenic differences among the species 42. Since SUDV

and BDBV have also shown their potential to cause public health emergencies during

several outbreaks in Central Africa, it is difficult to determine the priority for

development of countermeasure against those ebolaviruses.

In chapter I, I demonstrated generation of a broadly cross-reactive GP-specific

MAb. This MAb, 6D6, recognizes the putative epitope in the highly conserved IFL and

neutralizes infectivity of representative isolates of all known ebolavirus species by

inhibiting the membrane fusion. I further demonstrated its protective potential as a

therapeutic antibody in mouse models of EBOV and SUDV infections.

8

Materials and Methods

Viruses and cells. Variants Yambuku (EBOV1976), Makona (EBOV2014), Nzara

(SUDV), Butalya (BDBV), Pauléoula (TAFV), Philippines89 (RESTV) and Angola

(MARV), and mouse-adapted EBOV 43 were propagated in African green monkey kidney

Vero E6 cells and stored at -80 ˚C. Virus titters were determined as focus forming units

(FFU) by immunoplaque assays. All infectious work with filoviruses was performed in

the biosafety level 4 laboratories at the Integrated Research Facility of the Rocky

Mountain Laboratories, Division of Intramural Research, National Institute of Allergy

and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.

Replication-competent recombinant vesicular stomatitis virus (rVSV/EBOV GP,

rVSV/SUDV GP, and rVSV/RESTV GP) and replication-incompetent pseudotyped

vesicular stomatitis viruses (VSVs) containing green fluorescent protein (GFP) instead of

the VSV G gene were generated as described previously 20,44. Infectious units (IUs) of

replication-incompetent pseudotyped VSVs were determined using Vero E6 cells as

described previously 44. Vero E6 cells and human embryonic kidney (HEK) 293T cells

were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma). The media were

supplemented with fetal calf serum (FCS) (Cell Culture Bioscience) and 100 U/ml

penicillin, 0.1 mg/ml streptomycin (Gibco).

Purification of virus-like particles (VLPs) for immunization. HEK293T cells were

transfected with equal amounts of the expression plasmids encoding GP, matrix protein

(VP40), and nucleoprotein (NP) of EBOV or SUDV using TransIT LT-1 reagent (Mirus)

according to the manufacturer's instructions. Forty-eight hours later, the culture

supernatant was harvested and centrifuged at 3,500 rpm for 15 min to remove cell debris.

9

VLPs were purified from culture supernatants by ultracentrifugation at 28,000 rpm with

an SW32Ti rotor (Beckman) at 4 ˚C for 2 h with a 25% sucrose cushion. The VLP pellets

were suspended in phosphate-buffered saline (PBS) and fractionated through a 20-50%

sucrose gradient in PBS at 28,000 rpm with an SW41 rotor (Beckman) at 4 ˚C for 2 h.

Then the VLP fractions were diluted with PBS and sedimented by ultracentrifugation at

28,000 rpm with an SW41 rotor at 4 ˚C for 2 h. Finally, the VLP pellets were resuspended

in PBS.

Generation of MAb 6D6. Fifteen-week-old female BALB/c mice were immunized

intraperitoneally with 100 μg of EBOV VLPs. At 2 and 5 weeks after the first

immunization, the mice were intraperitoneally immunized with 100 μg of EBOV VLPs.

At 10 weeks after the first immunization, the mice were immunized with 100 μg of SUDV

VLPs. Two weeks after the last immunization, the mice were boosted intraperitoneally

with 100 μg of EBOV VLPs. Three days later, the mice were euthanized and spleen cells

and mouse myeloma P3U1 cells were fused and maintained according to a standard

procedure45. The mice were treated daily with 75 µg/kg rapamycin intraperitoneally

starting 1 week prior to the primary immunization until euthanasia. Hybridomas were

screened for secretion of EBOV GP-specific MAbs by a neutralization test with VSV

pseudotyped with EBOV GP and hybridomas producing MAbs were cloned twice by

limiting dilution of the cells. Hybridomas producing neutralizing MAbs were further

screened for the cross-reactivity to the other filovirus GPs. MAb 6D6 (IgG1) was found

to be a broadly cross-neutralizing MAb and was purified from mouse ascites using protein

A agarose columns (Bio-Rad). Animal studies were carried out in strict accordance with

the Guidelines for Proper Conduct of Animal Experiments of the Science Council of

10

Japan. The protocol was approved (13-0136) by the Hokkaido University Animal Care

and Use Committee.

Neutralization tests. To evaluate the neutralizing activity a focus forming assay was used.

EBOV, SUDV, TAFV, BDBV, RESTV, and MARV was appropriately diluted to yield 20-

100 FFUs and mixed with purified MAb 6D6 for 1 h at 37 ˚C and inoculated into

confluent Vero E6 cells grown in 96-well tissue culture plates. After incubation for 3 days,

the cells infected with filoviruses were fixed and stained with a mixture of anti-GP (mouse

anti-EBOV GP ZGP42/3.7 or anti-MARV GP rabbit serum FS0505) and anti-NP (mouse

anti-EBOV NP ZNP74/7 or anti-MARV NP rabbit serum FS0609) primary antibodies 46

followed by goat anti-mouse IgG/Alexa Fluor 488 (Invitrogen) and goat anti-rabbit

IgG/Alexa Fluor 488 (Invitrogen) secondary antibodies. Virus infectivity was quantified

by counting the number of fluorescent foci. VSV pseudotyped with filovirus GPs were

appropriately diluted to yield 300 to 1,500 IUs and mixed with purified MAb 6D6,

ZGP133/3.16, or ZGP226/8.1 for 1 h at room temperature, and inoculated into confluent

Vero E6 cells grown in 96-well plates. At 20 h post-inoculation, GFP-positive cells were

counted using IN Cell Analyzer 2000 (GE Healthcare). To reduce the background

infectivity of the parent VSV G, pseudotyped viruses were treated with a neutralizing

MAb to VSV G protein (VSV-G[N]1-9) before use. The relative percentage of infectivity

was calculated by setting the number of cells infected in the absence of MAb 6D6 to

100%.

Enzyme-linked immunosorbent assay (ELISA). GP-based ELISA was performed as

described previously 42. Soluble forms of EBOV GP were purified and used as antigens.

11

MAbs were serially diluted with PBS containing 0.05% Tween 20 and 1% skim milk.

Bound antibodies were visualized with horseradish peroxidase-conjugated goat anti-

mouse IgG (H+L) (Jackson ImmunoResearch) and 3,3’,5,5’-tetramethylbenzidine

(Sigma). The reaction was stopped by adding 1 M phosphoric acid, and the optical density

at 450 nm (OD450) was measured using SoftMax Pro 6.2.1 software (Molecular Devices).

Selection of escape mutants and identification of the putative epitope. Tenfold serial

dilutions of rVSV/EBOV GP, rVSV/SUDV GP, and rVSV/RESTV GP were incubated

with 10 μg/ml MAb 6D6 for 1 h at room temperature and inoculated into confluent Vero

E6 cells grown in 6-well tissue culture plates. After adsorption for 1 h, the cells were

overlaid with Eagle’s minimal essential medium (Invitrogen) containing 0.8% Bacto Agar

(BD), 0.3% bovine serum albumin (BSA) (Sigma), 100 U/ml penicillin, 0.1 mg/ml

streptomycin, and 10 μg/ml 6D6, and then incubated for 2 days at 37 ˚C. Mutant viruses

growing in the presence of MAb 6D6 were purified from single isolated plaques at the

highest dilution of the virus and propagated in Vero E6 cells. Viral RNAs were extracted

from the supernatant, the nucleotide sequences of the GP genes of the parent viruses and

the escape mutants were determined. The deduced amino acid sequences were compared

among these viruses. The IFL amino acid sequences of EBOV, SUDV, BDBV, TAFV,

RESTV, and MARV were obtained from GenBank (Accession numbers, U23187.1,

U28134.1, NC_014373.1, U28006.1, AF522874.1, and KY047763, respectively). The

substituted amino acid positions were mapped on the trimeric structure of GPs

constructed using Discovery Studio 4.1 (Biovia) based on the crystal structure of EBOV

GP (PDB code: 3CSY). The SUDV and RESTV GP structures were generated by

homology modelling based on the EBOV GP structure. From 100 models of the GP trimer,

12

the model with the best score for probability density function (PDF) and total energy was

chosen. The model was evaluated using Profiles-3D 47.

Purification and fluorescent-labeling of VLPs. For purification of VLPs, equal

amounts of the expression plasmids for EBOV GP, VP40, and NP were transfected into

HEK293T cells by using TransIT LT-1 (Mirus). Forty-eight hours post-transfection, the

culture supernatant was harvested and centrifuged at 3,500 rpm for 15 min to remove cell

debris. VLPs were precipitated through a 25% sucrose cushion by centrifugation at 11,000

rpm for 1 h at 4 ˚C with an SW32Ti rotor (Beckman). Precipitated VLPs were suspended

in PBS, and fractionated through a 20-50% sucrose gradient in PBS at 27,000 rpm with

an SW41 rotor (Beckman) for 2.5 h at 4 ˚C. One ml of fractionated VLPs (1 μg/ml) was

incubated with 0.6 µl of 100 µM stock solution of 1,1'-dioctadecyl-3,3,3',3'-

tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen) in the dark for 1 h at room

temperature with gentle agitation 11.

Imaging of attachment, internalization and membrane fusion of DiI-labeled VLPs.

Vero E6 cells expressing enhanced GFP fused to Rab7 (eGFP-Rab7) were cultured in 35

mm glass-bottom culture dishes (MatTek Corporation). DiI-labeled VLPs were treated

with 20 μg/ml 6D6 or control IgG (CTR IgG) (mouse IgG1,κ; BD Biosciences) for 1 h at

room temperature. The cells were then washed with 1 ml of phenol red-free DMEM

(Invitrogen) and incubated with either MAb 6D6-treated, CTR IgG-treated or untreated

VLPs in the same medium on ice for 30 min. Following this, the cells were washed with

the same medium to remove unbound VLPs and incubated with 200 µl of phenol red-free

DMEM containing 2% FCS and 4% BSA at 37 ˚C for 0, 2, and 6 h to analyze attachment,

13

internalization and membrane fusion, respectively. In this assay, the fluorescent signal is

enhanced once the DiI-labeled VLP envelope fuses with the endosomal membrane 11. To

count the number of DiI-labeled VLPs, the cells were fixed in 4% paraformaldehyde for

15 min at room temperature. Then nuclei were stained using 1 μg/ml of 4',6-diamidino-

2-phenylindole, dihydrochloride (DAPI) for 10 min at room temperature (Thermo Fisher

Scientific). Images were acquired with a 63 × oil objective lens on a Zeiss LSM700

inverted microscope and ZEN 2009 software (Carl Zeiss). For measurement of the

number of DiI-labeled VLPs, images of 4-20 optical sections were acquired in 0.5-1

micron steps. The number of DiI signals was determined in approximately 100 individual

cells (approximately 1-20 dots/cell) and the average number per cell was calculated for

each condition. For colocalization analysis, the percentage of DiI-labeled VLPs that

colocalized with eGFP-Rab7 (Both DiI- and eGFP-positive pixels/DiI-positive pixels ×

100) was measured using the Coloc module in ZEN 2010 software (Carl Zeiss). The

number, size, and fluorescence intensity of DiI signals were analyzed with MetaMorph

software (Molecular Devices). The relative sizes and intensities of DiI signals were

determined by defining the value of untreated cells as 1.

Passive immunization and protective efficacy in mice. BALB/c mice (female, 6-8

weeks old) were inoculated with mouse-adapted EBOV (1,000 FFU) by intraperitoneal

injection in a total volume of 200 μl. One day after infection, the mice were treated with

100 μg of MAb 6D6 intraperitoneal in a volume of 200 μl. To investigate the potential of

6D6 to protect mice from EBOV and SUDV infection, C57BL/6 IFNAR-/- mice were

chosen for this study as they are known to be susceptible to EBOV and SUDV infection.

BDBV did not cause clinical symptoms in IFNAR-/- mice (data not shown). IFNAR-/- mice

14

(male and female, 5-8 weeks old) were treated with 100 μg of MAb 6D6 intraperitoneal

in a volume of 200 μl one day after infection with EBOV1976 (1,000 FFU) or SUDV

(1,000 FFU). The animals were monitored for signs of illness and weighed daily.

Surviving mice were euthanized 28 days after infection, and serum was collected for

serology. Research was approved and conducted in compliance with the guidelines of the

National Institute of Allergy and Infectious Diseases/Rocky Mountain Laboratories

Institutional Animal Care and Use Committee. The facility where this research was

conducted is fully accredited by the Association for the Assessment and Accreditation of

Laboratory Animal Care International and has an approved Office of Laboratory Animal

Welfare Assurance (#A4149-01). All procedures were conducted by trained personnel

under the supervision of veterinarians, and all invasive clinical procedures were

performed while animals were anesthetized. Early endpoint criteria, as specified by the

Institutional Animal Care and Use Committee approved scoring parameters, were used to

determine when animals should be humanely euthanized.

Statistical analysis. All data were analyzed using the GraphPad Prism v6.0 software. For

the viral attachment, internalization, and membrane fusion experiments, Student’s t-test

was used to evaluate differences between 6D6 and CTR IgG. To assess the weight loss of

mice, I performed a 2-way repeated-measures analysis of variance (ANOVA), followed

by multiple t-tests comparing the average weights of 6D6-treated and untreated (control)

mice at each time point, using the Holm–Sidak method. P values less than 0.05 were

considered to be statistically significant.

15

Results

In vitro properties of MAb 6D6. The cross-reactive MAb 6D6 was selected by screening

mouse hybridoma supernatants thoroughly for the cross-neutralizing activity of GP-

specific MAbs. MAb 6D6 was found to be GP-specific and to efficiently neutralize the

infectivity of VSV pseudotyped with GPs of all known ebolavirus species (EBOV, SUDV,

TAFV, BDBV, and RESTV), including the variant that caused the latest outbreak in West

Africa (EBOV2014), but not Marburg virus (MARV) which a related filovirus and causes

human disease similar to EVD (Fig. 1A). The 50% inhibitory concentrations (IC50) of

6D6 for VSVs bearing EBOV1976, EBOV2014, SUDV, TAFV, BDBV, and RESTV GPs

were 0.05, 0.12, 0.19, 0.33, 0.24, and 0.62 μg/ml, respectively. I then confirmed that 6D6

effectively neutralized the infectivity of representative authentic isolates of all known

ebolavirus species (Fig. 1B). Furthermore, binding experiments to EBOV GP and

neutralization assays with EBOV GP-pseudotyped VSV revealed that 6D6 possessed

higher binding and neutralizing abilities than EBOV GP-specific MAbs ZGP133/3.16 and

ZGP226/8.1 (Fig. 1C and D), which have shown promising protective efficacy in animal

models of lethal EBOV infection 28,48.

Identification of the putative 6D6 epitope. To determine the putative epitope of MAb

6D6, I utilized replication-competent recombinant VSV containing the EBOV, SUDV, or

RESTV GP gene 20. The putative epitopes of ZGP133/3.16 and ZGP226/8.1 have been

successfully determined by identifying the amino acid substitutions observed in the

antigenic variants escaping from neutralization by the antibodies 20,24. I cloned 6 escape

mutants of EBOV GP and found that each mutant had a single amino acid substitution,

Gly-to-Arg (5/6) or Gly-to-Glu (1/6), at amino acid position 528 within the IFL sequence

16

Fig. 1. Neutralizing properties of MAb 6D6 against ebolaviruses. (A) VSV

pseudotyped with the indicated GPs or (B) infectious EBOV, SUDV, TAFV, BDBV,

RESTV, and MARV were incubated with purified MAb 6D6 followed by inoculation into

confluent Vero E6 cells. (C) Binding activities of MAbs 6D6 (red), ZGP133/3.16 (orange)

and ZGP226/8.1 (blue) were examined by ELISA using EBOV GP as the antigen. (D)

Neutralizing activities of MAbs 6D6 (red), ZGP133/3.16 (orange), and ZGP226/8.1

(blue) against VSV pseudotyped with EBOV GP are shown. The mean and standard

deviation of three independent experiments are shown.

17

in the GP2 subunit (Fig. 2A). One of the six SUDV GP escape mutants had a Gly-to-Arg

substitution at position 528, and other 5 SUDV GP escape mutants had an Ala-to-Thr

substitution at position 530 (Fig. 2A). Two of the six RESTV GP escape mutants had a

Gly-to-Glu substitution at position 529, which corresponded to position 528 of EBOV GP.

A total of 3 amino acid changes were found in the other 4 RESTV GP escape mutants

(Fig. 2A). Using a reverse genetics approach I verified that the Leu-to-Trp substitution at

position 530 was critical for escape from 6D6 neutralization (Fig. 3). These amino acid

positions, which are located at the tip of the IFL structures of EBOV, SUDV, and RESTV

GPs, indicate that the loop structure including these residues is important to form the

recognition site of 6D6 (Fig. 2B). I confirmed that 6D6 did not bind to the chimeric Ebola

GP whose IFL region was replaced with that of MARV; however, 6D6 showed no binding

activity to the synthetic peptide corresponding to the amino acids of the IFL of EBOV GP

(not shown), suggesting that the 6D6 epitope may partly include other conformational

structures. Importantly, the amino acid sequence of the IFL region is highly conserved

among all currently known ebolaviruses (Fig. 2A), providing a novel target for universal

antibody therapy against EVD caused by human-pathogenic ebolaviruses (EBOV, SUDV,

TAFV, and BDBV).

Mechanism of the neutralizing activity of 6D6. Since the IFL is crucial for GP-mediated

membrane fusion, I assumed that 6D6 directly inhibited the fusion step during the entry

process of ebolaviruses into cells. To confirm this, I analyzed the inhibitory effects of 6D6

on viral attachment, internalization, and membrane fusion using DiI-labeled VLPs 11. The

number of 6D6-treated VLPs attached to the surface of Vero E6 cells was not significantly

different from that of untreated or CTR IgG-treated VLPs, indicating that 6D6 did not

18

Fig. 2. Identification of the putative epitope of MAb 6D6. (A) Structure of GP and

amino acid sequences of the internal fusion loop (IFL). The GP1 subunit contains the

receptor binding domain (RBD), a glycan cap and a mucin-like domain (MLD). The GP2

subunit contains the IFL, heptad repeats 1 and 2 (HR1 and HR2), the transmembrane

region (TM), and the cytoplasmic tail (CT). Amino acid substitutions found in the EBOV,

SUDV, and RESTV GP escape mutants selected under MAb 6D6 pressure are shown in

19

red. (B) The amino acid residues (green, blue, and yellow spheres represent Gly, Ala, and

Leu, respectively) critical for escape from the MAb 6D6 neutralization are mapped on the

trimeric structure of GPs. GP1 (blue) and GP2 (red) monomers are shown as ribbon

models.

20

Fig. 3. Identification of the key amino acid residue on the escape mutant RESTV GP

selected by MAb 6D6. Two RESTV GP mutants had the same Gly-to-Glu substitution at

position 529, which is the corresponding position of the EBOV GP mutants, whereas three

amino acid changes were found in the other 4 mutants of RESTV GP: Tyr at position 517,

Val at position 522, and Leu at position 530 were replaced with His, Ala, and Trp,

respectively (Fig. 2B). To clarify which amino acid change was critical for escaping from

the 6D6 neutralization, I generated RESTV GP mutants with single amino acid

substitutions for each position (Y517H, V522A, and L530W), and investigated the

neutralizing activity of MAb 6D6 against VSV pseudotyped with these single amino acid

mutants of RESTV GP. The mean and standard deviation of three independent

experiments are shown.

21

interfere with VLP attachment (Fig. 4A and D). Likewise, the number of VLPs that

colocalized with eGFP-Rab7, a late endosome marker, was similar, suggesting that 6D6

did not affect subsequent uptake into cells (Fig. 4B and E). Finally, I analyzed membrane

fusion efficiency by detecting dequenched DiI fluorescence 11,46. I observed remarkably

enlarged and enhanced DiI signals colocalizing with Rab7 in cells incubated with

untreated and CTR IgG-treated VLPs, indicating that membrane fusion occurred

efficiently in the endosomes (Fig. 4C left and middle panels). In contrast, the size and

intensity of DiI signals from 6D6-treated VLPs were significantly reduced, indicating that

6D6 prevented GP-mediated membrane fusion (Fig. 4C right panels and F).

Protective efficacy of MAb 6D6 in mouse models. Finally, I investigated the potential

of 6D6 to protect mice from ebolavirus infections (Fig. 5). Immunocompetent BALB/c

mice were infected with a lethal dose of mouse-adapted EBOV and treated 24 h later with

100 μg of 6D6. The treated animals survived without clinical symptoms, whereas

untreated mice succumbed to infection within 9 days (Fig. 5A). I further evaluated the

cross-protective potential of 6D6 against wild-type EBOV and SUDV infections (Fig.

5B). Since immunocompetent mice do not develop disease upon infection with these

wild-type ebolavirus isolates, interferon α/β receptor knockout (IFNAR-/-) C57BL/6 mice

were used for this purpose. I found that both EBOV and SUDV caused severe weight loss

in untreated mice, whereas only EBOV uniformly caused a lethal infection. Treatment

with 6D6 24 h after infection delayed the onset of the disease caused by these ebolaviruses

and significantly reduced the weight loss in this immunocompromised mouse strain. All

6D6-treated mice survived the EBOV infection.

22

23

Fig. 4. Effect of MAb 6D6 on virus entry. (A-C) Fluorescent images of attachment and

internalization of VLP. (D-F) Quantified fluorescent signals of VLP and Rab7. Untreated

(Control), CTR IgG (CTR IgG)- or 6D6-treated DiI-labeled VLPs were inoculated into

confluent Vero E6 cells expressing eGFP-Rab7 and incubated for 30 min on ice. After

adsorption, the cells were incubated for 0 (A and D), 2 (B and E) and 6 h (C and F) at 37

˚C. DiI signals on the cell surface (A) and in the cytoplasm (B and C) were monitored by

24

confocal laser scanning microscopy. The number, size, and fluorescence intensity of DiI

signals were quantified. Scale bars represent 10 µm. The means and standard deviations

of three independent experiments are shown. Statistical analysis was performed using

Student’s t-test (*p<0.05).

25

Fig. 5. Protective efficacy of MAb 6D6 in mice. (A) BALB/c mice (n = 8) were

intraperitoneally infected with a lethal dose of mouse-adapted EBOV. (B) IFNAR-/-

mice

(n = 6) were intraperitoneally infected with EBOV1976 (upper panels) or SUDV (bottom

panels). Twenty-four hours after infection, animals were treated intraperitoneally with

either 100 μg of MAb 6D6 or a vehicle control (PBS). The animals were then monitored

for 14 days for clinical signs of infection and weighed daily. Body weight (left panels)

and survival curves (right panels) are shown. Error bars indicate standard error of the

mean. Significant differences are indicated by asterisks (*p<0.05).

26

Discussion

Neutralizing antibody-based therapies have been tested in animal models and

clinical trials with particular attention given to highly lethal viral diseases 49. Passive

immunization with convalescent serum has also been tested for EBOV-infected patients

50,51. Recent studies have demonstrated the effectiveness of MAb treatments in nonhuman

primate models of EVD 26,28-30,52,53, and GP-specific MAb cocktails, ZMapp and ZMAb,

were used in clinical cases during the 2014 EVD outbreak caused by EBOV (belonging

to Zaire ebolavirus) 32-35. However, these MAb cocktails are not expected to be cross-

protective against the other antigenically distinct ebolaviruses. On the other hand, highly

cross-reactive MAbs against all known ebolaviruses have been generated previously, but

none of those has neutralizing activity 42,54. In this study, I generated the novel MAb 6D6,

which has cross-neutralizing activity against all known ebolaviruses.

I showed that 6D6 reduced infectivities of all known ebolaviruses in vitro with

higher neutralizing and binding activities than the EBOV GP-specific MAbs

ZGP133/3.16 and ZGP226/8.1. Indeed, the IC50 values of 6D6 were equal to or lower

than those of previously reported neutralizing MAbs 55-57, suggesting its protective

capacity as a therapuetic MAb. By analyzing the amino acid substitution observed in the

antigenic variants escaping from 6D6, I determined the putative epitope of 6D6 in the IFL

on the GP molecule, which may overlap that of a partially cross-reactive GP-specific

MAb reported recently 58. Accordingly, 6D6 directly inhibited the membrane fusion

induced by EBOV VLPs in endosomes/lysosomes. The IFL structure is highly conserved

in all species of ebolaviruses, indicating that this region can be targeted for both vaccine

and therapeutic development against ebolaviruses. Since the putative epitope of 6D6 is

different from those previously reported for other GP-specific MAbs used in passive

27

immunization studies 20,56,59-62. 6D6 may provide a promising option as a component of

antibody cocktails in combination with other previously tested MAbs.

I further demonstrated that passive immunization with 6D6 protected both

BALB/c and IFNAR-/- C57BL/6 mice from lethal infection by EBOV (i.e., mouse-

adapted and wild-type EBOV, respectively). However, the cross-protective potential of

6D6 could only be evaluated for disease severity using IFNAR-/- C57BL/6 mice since

mouse-adapted SUDV causing lethal infection in immunocompetent mice is not currently

available and SUDV did not uniformly cause lethal infection even in this

immunocompromised mouse strain. I found that passive immunization with 6D6

significantly reduced the weight loss in SUDV-infected mice, although the extent was not

as prominent as in EBOV-infected mice. The less significant protective effects seen in

SUDV-infected mice might have been due to the higher IC50 value of 6D6 against SUDV

than against EBOV. Thus, these results supported the in vitro characteristics of 6D6 and

demonstrated the effectiveness of the 6D6 treatment in vivo against multiple species of

ebolaviruses. A previous study demonstrated that immunocompromised mice treated

several times with 500 μg of MAb SUDV-specific MAbs were protected from SUDV

infection 63, whereas mice were treated once with 100 μg of 6D6 in this study. Thus, I

assume that the protective effect against SUDV could be improved by increased doses of

6D6.

The broadly cross-neutralizing antibody 6D6 recognizing the common epitope

shared among all currently known ebolaviruses, converted into a human-mouse chimeric

MAb 28, is a promising therapeutic candidate. On the other hand, the generation of 6D6

escape mutants in vitro speaks against monotherapy with this MAb and may favor the

development of antibody cocktails including 6D6 for future pan-ebolavirus therapy. For

28

other viruses, it has indeed been reported that combination of MAbs helps to avoid the

appearance of escape variants if these MAbs recognize distinct epitopes 64-66. While the

detailed mechanisms underlying antibody-mediated protection from ebolavirus infection

need to be further elucidated, the discovery of this highly cross-reactive neutralizing

antibody and its putative epitope reported here provides a promising option for the

development of a universal EVD therapy and will accelerate the design and

implementation of improved therapeutics and vaccines that can selectively elicit cross-

neutralizing antibodies against multiple species of ebolaviruses.

29

Summary

During the latest outbreak of EVD in West Africa, MAb therapy (e.g., ZMapp)

was utilized to treat patients. However, due to the antigenic differences among the five

ebolavirus species, the current therapeutic MAbs are only effective against viruses of the

species Zaire ebolavirus. Although this particular species has indeed caused the majority

of human infections in Central and, recently, West Africa, other ebolavirus species (e.g.,

Sudan ebolavirus and Bundibugyo ebolavirus) have also repeatedly caused outbreaks in

Central Africa and thus should not be neglected in the development of countermeasures

against other ebolavirus species. Here I report the generation of an ebolavirus GP-specific

MAb that effectively inhibits cellular entry of representative isolates of all known

ebolavirus species in vitro and show its protective efficacy in mouse models of ebolavirus

infections. This novel neutralizing MAb targets a highly conserved IFL in the GP

molecule and prevents membrane fusion of the viral envelope with cellular membranes.

The discovery of this highly cross-neutralizing antibody provides a promising option for

broad-acting ebolavirus antibody therapy and will accelerate the design of improved

vaccines that can selectively elicit cross-neutralizing antibodies against multiple species

of ebolaviruses.

30

Chapter II:

Fcγ-receptor IIa-mediated Src signaling pathway is essential for the

antibody-dependent enhancement of Ebola virus infection

Introduction

It has been demonstrated that EBOV exploits some GP-specific antibodies for

its entry into cells, leading to increased infectivity in vitro 22,39. This phenomenon has

been described for a number of viruses and is known as ADE 36-38. For some of these

viruses, ADE has become a great concern to disease control by vaccination. Particularly,

convalescent human sera have been shown to contain ADE antibodies 22,39, raising

concerns about potential detrimental effects of passive immunization with convalescent

human sera, which is currently under consideration for treatment of EVD. Two distinct

pathways of EBOV ADE, one mediated by Fc receptors and the other by complement

component C1q and its ligands, are known 22,23. In particular, the FcγR is commonly

involved in ADE of virus infections 67,68. However, the molecular mechanisms underlying

ADE-mediated virus entry through FcγR are not fully understood.

Three classes of FcγR, FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), are

expressed in various human immune cells such as dendritic cells, monocytes, and B

lymphocytes 69. Among these FcγRs, FcγRII is a key molecule for EBOV ADE of

infection in human leukemia K562 cells 23. Human FcγRII exists in two isoforms, FcγRIIa

and FcγRIIb, which differ in their signal peptides and cytoplasmic tails. FcγRIIa is the

active form of FcγRII and contains an immunoreceptor tyrosine-based activation motif

(ITAM) in its cytoplasmic tail 69. The cytoplasmic tail of FcγRIIa is known to contribute

to the activitation of two structurally and functionally distinct protein-tyrosine kinase

31

(PTK) classes, the sarcoma (Src) family PTKs 70,71 and spleen tyrosine kinase (Syk) 72. In

addition, Syk is reported to participate in activation of enzymes such as rat sarcoma (Ras),

phosphatidylinositol 3-kinase (PI3K), and Bruton’s tyrosine kinase (Btk) 69,73. These

signaling pathways are known to be important for the induction of phagocytic and

endocytic processes to internalize immune complexes 69,73,74.

In the chapter II, I focused on the role of FcγRIIa and investigated the

contribution of FcγRIIa-mediated signaling to the ADE of EBOV infection. I show that

Src family PTKs are essential for EBOV ADE-mediated entry. My data indicate that

binding of antibody-virus complexes to the cell surface FcγRIIa triggers phosphorylation

of Src family PTKs and activates subsequent signaling pathways, leading to enhanced

viral uptake through phagocytosis and/or macropinocytosis.

32

Materials and Methods

Viruses and cells. EBOV expressing GFP 75 was propagated in Vero E6 cells and stored

at -80 ˚C. Replication-incompetent VSV pseudotyped with EBOV GP containing GFP

instead of the VSV G gene (VSV-EBOV GP) was generated as described previously 20,44.

Virus titers were determined as IUs by counting GFP-positive cells. All infectious work

with EBOV was performed in the biosafety level 4 laboratory at the Integrated Research

Facility of the Rocky Mountain Laboratories, Division of Intramural Research, National

Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton,

Montana, USA. Vero E6 cells and HEK 293T cells were grown in DMEM (Sigma), and

human chronic myelogenous leukemia K562 cell line and K562 cell lines stably

expressing dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) or human

macrophage galactose-type C-type lectin (hMGL) 76,77 and human leukemic Jurkat T cells

were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma). These

media were supplemented with 10% FCS (Cell Culture Bioscience), 100 U/ml penicillin,

and 0.1 mg/ml streptomycin (Gibco).

Generation of FcγRIIa- or FcγRIIaΔCT-expressing Jurkat T cells. The FcγRIIa gene

was PCR-amplified from a full-length cDNA library prepared from K562 cells using the

primers, EcoRI-FcγRIIa (5’-GGGAATTCGGATGACTATGGAGACCCAA-3’) and

FcγRIIa-XhoI (5’-ATTTCTCGAGTTTGTCATCCACTCAGCAAG-3’). Mutant FcγRIIa

lacking its cytoplasmic tail (amino acid positions 241-317) (FcγRIIaΔCT) was generated

using a PrimeSTAR Mutagenesis Basal Kit (Takara). After sequence confirmation, these

PCR products were cloned into a murine leukemia virus-based retroviral vector, pMXs-

Puro Retroviral Vector (Cell Biolabs). To generate the retrovirus, 293T-derived Platinum-

33

GP (Plat-GP) cells (Cell Biolabs) were cotransfected with pMXs-puro encoding FcγRIIa

or FcγRIIaΔCT and the expression plasmid pCAGGS encoding the VSV G protein using

Lipofectamine 2000 (Invitrogen). Forty-eight h later, the culture supernatants containing

retroviruses were collected, clarified through 0.45 μm filters, and then used to infect

Jurkat T cells. Jurkat T cell lines stably expressing FcγRIIa or FcγRIIaΔCT were selected

with RPMI medium containing 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin,

and 10 µg/ml puromycin (Sigma-Aldrich). For some experiments, each Jurkat T cell line

was cloned by limiting dilution to enrich the population of FcγRIIa-expressing cells. To

check the expression levels of FcγRIIa and FcγRIIaΔCT, these cells were incubated with

a mouse anti-CD32 monoclonal antibody (GeneTex) for 1 h at room temperature. After

washing 3 times with PBS, binding of the primary antibody was detected with Alexa647-

conjugated F(ab')2-goat anti-mouse IgG (H+L) (Jackson ImmunoResearch). After further

washing 3 times with PBS, the fluorescent intensity of the cells was analyzed using a

FACS Canto flow cytometer (BD Biosciences) and FlowJo software (Tree Star).

ADE assays. EBOV was appropriately diluted to yield 50-100 IUs in K562 cells and then

incubated for 30 min-1 h at 37 ˚C with or without 10 μg/ml MAbs. The anti-EBOV GP

MAb ZGP12/1.1 (IgG2a), which is known to enhance EBOV infection in vitro, was used

as the ADE MAb 39. S139/1 (IgG2a), a MAb specific to influenza A virus hemagglutinin,

was used as the control IgG (CTR IgG) 78. K562 and Jurkat T cells were inoculated with

EBOV alone or EBOV/MAb mixtures and incubated for 72 h. Virus infectivity was

measured by counting the number of GFP-positive cells in FACS and analyzed using

FlowJo software. VSV-EBOV GP was appropriately diluted to yield 50-100 IUs in K562

cells and incubated for 1 h at room temperature with or without 1 μg/ml MAbs, and then

34

inoculated into K562 and Jurkat T cells. Twenty-four h later, GFP-positive cells were

counted using an IN Cell Analyzer 2000 (GE Healthcare). To reduce the background (i.e.,

residual) infectivity of the parent VSV, VSV-EBOV GP was treated with a neutralizing

MAb to VSV G protein (VSV-G[N]1-9) before use.

Purification and DiI-labeling of VLPs. For purification of VLPs, HEK293T cells were

transfected with equal amounts of the expression plasmids encoding EBOV or SUDV GP,

VP40, and NP using TransIT LT-1 (Mirus) according to the manufacturer's instructions.

Forty-eight h after transfection, the culture supernatant was harvested and centrifuged at

3,500 rpm for 15 min at 4 ˚C to remove cell debris. VLPs were precipitated through a

25% sucrose cushion by centrifugation at 11,000 rpm for 1 h at 4 ˚C with an SW32Ti

rotor (Beckman). Pelleted VLPs were suspended in PBS, and fractionated through a 20-

50% sucrose gradient in PBS at 28,000 rpm with an SW41 rotor (Beckman) for 2 h at 4

˚C. One ml of 1 μg/ml fractionated VLPs was incubated with 0.6 µl of 100 µM DiI

(Molecular Probes) in the dark for 1 h at room temperature with gentle agitation 11.

Imaging of attachment and internalization of DiI-labeled VLPs. The eGFP-Rab7

fusion protein gene was cloned into a Moloney murine leukemia virus-based retrovirus

plasmid 11,79, and recombinant retroviruses for the expression of eGFP-Rab7 were

produced and used to infect K562 cells as described above. K562 and Jurkat T cell lines

were cultured in 35 mm glass-bottom dishes (MatTek Corporation) precoated with borate

buffer containing 0.1 mg/ml poly-L-lysine (Sigma). The cells were washed with phenol

red-free RPMI (Gibco) and inoculated with 100 μl of 1 μg/ml DiI-labeled VLPs treated

with 20 μg/ml ZGP12/1.1 or CTR IgG for 1 h at room temperature, followed by

35

incubation for 30 min on ice. They were then washed twice with the same medium to

remove unbound DiI-labeled VLPs and incubated with phenol red-free RPMI containing

2% FCS and 4% BSA for 0 and 2 h at 37 ˚C to analyze DiI-labeled VLP attachment and

internalization, respectively. To count the number of DiI-labeled VLPs, the cells were

fixed with 4% paraformaldehyde for 15 min at room temperature. Then the nuclei were

stained with 1 μg/ml DAPI (Molecular Probes) for 10 min at room temperature.

Microscopic images were acquired with a 63 × oil objective lens on a Zeiss LSM780

inverted microscope and ZEN 2010 software (Carl Zeiss). For measurement of the

number of DiI-labeled VLPs, images of 4-20 optical sections were acquired in 1 micron

steps. The number of DiI-labeled VLPs was determined in approximately 100 individual

cells using MetaMorph software (Molecular Devices) and the average number per cell

was calculated for each condition. For colocalization analysis, the percentage of DiI-

labeled VLPs that colocalized with eGFP-Rab7 (Both DiI- and eGFP-positive pixels/DiI-

positive pixels × 100) was measured in approximately 100 individual cells using the

Coloc module in ZEN 2010 software (Carl Zeiss).

Dextran uptake assays. One μg/ml DiI-labeled VLPs were treated with 20 μg/ml CTR

IgG or ZGP12/1.1 for 1 h at room temperature. K562 and Jurkat T cell lines were cultured

in poly-L-lysine-coated glass-bottom culture dishes and incubated with 100 μl untreated,

CTR IgG-, or ZGP12/1.1-treated DiI-labeled VLPs for 30 min on ice. The cells were

washed twice with phenol red-free RPMI and then incubated with phenol red-free RPMI

containing 2% FCS, 4% BSA, and 0.5 mg/ml Dextran, Alexa Fluor 647, 10,000 MW

(Alexa647-labeled Dx10) (Molecular Probes) for 1-2 h at 37 ̊ C. After washing twice with

phenol red-free RPMI to remove surface-unbound DiI-labeled VLPs and Alexa647-

36

labeled Dx10, and the cells were fixed with 4% paraformaldehyde for 15 min at room

temperature. Then, the nuclei were stained with 1 μg/ml DAPI for 10 min at room

temperature. Internalized DiI-labeled VLPs and Alexa647-labeled Dx10 were analyzed

by confocal laser scanning microscopy as described above. The percentage of DiI-labeled

VLPs that colocalized with Alexa647-labeled Dx10 (Both DiI- and Alexa647-positive

pixels/DiI-positive pixels × 100) was measured in approximately 100 individual cells

using the Coloc module in ZEN 2010 software. The number and size of Dx10-positive

vesicles were analyzed with MetaMorph software.

Inhibitor treatments. For infection assays, the Syk inhibitor R788 (Santa Cruz), Src

family PTK inhibitor PP2 (Tocris), BTK inhibitor LFM-A13 (Focus Biomolecules), PI3K

inhibitor LY294002 (Wako), and Ras inhibitor Manumycin A (Santa Cruz) were used for

treatments of K562 cells. R788, LFM-A13, and LY294002 were used at 0.15-40 μM. PP2

and Manumycin A were used at 0.15-10 μM. For imaging analysis, K562 cell lines were

cultured in 35 mm glass-bottom dishes precoated with poly-L-lysine, and then treated

with 20 μM PP2 for 1 h at 37 ˚C. PP2-treated cells were washed with phenol red-free

RPMI and inoculated with untreated, CTR IgG-, or ZGP12/1.1-treated DiI-labeled VLPs

for 30 min on ice in the presence of 20 μM PP2 in the same medium. The cells were then

washed twice with the same medium and incubated with phenol red-free RPMI containing

2% FCS, 4% BSA, and 20 μM PP2 for 0 and 2 h at 37 ˚C. Then they were fixed and

analyzed by confocal laser scanning microscopy as described above. Dimethyl sulfoxide

(DMSO, Sigma-Aldrich) or ethanol (Kanto Chemical) was used as a solvent control.

Generation of Src or Syk knockdown K562 cells. Plat-GP cells were cotransfected with

37

pRS (retroviral plasmids) encoding human Src or Syk shRNA (ORIGENE) and the

expression plasmid pCAGGS encoding the VSV G protein using Lipofectamine 2000

(Invitrogen). ShSrc target sequences were: shSrc1:5’-

GGAGGCTTCAACTCCTCGGACACCGTCAC-3’, shSrc2: 5’-

AAGAAAGGCGAGCGGCTCCAGATTGTCAA-3’, shSrc3: 5’-

GCAGTTGTATGCTGTGGTTTCAGAGGAGC-3’, shSrc4: 5’-

CTGGAGGCAATCAAGCAGACATAGAAGAG-3’. ShSyk target sequences were:

shSyk1:5’- GAATATGTGAAGCAGACATGGAACCTGCA-3’, shSyk2: 5’-

GGAGGAGGCAGAAGATTACCTGGTCCAGG-3’, shSyk3: 5’-

TGTCATTCAATCCGTATGAGCCAGAACTT-3’, shSyk4: 5’-

CTCTGGCAGCTAGTCGAGCATTATTCTTA-3’. After incubation for 48 h, culture

supernatants containing the retroviruses expressing human Src or Syk shRNAs were

collected, clarified through 0.45-μm filters, and then used to infect K562 cells.

Transduced K562 cell lines were selected with RPMI medium containing 10% FCS, 100

U/ml penicillin, 0.1 mg/ml streptomycin, and 5 µg/ml puromycin (Sigma-Aldrich). To

check the knockdown efficiency for Src and Syk, cells were collected and washed once

with PBS and treated with lysis buffer (0.1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA,

10 mM Tris HCl, pH 7.8) in the presence of a protease inhibitor cocktail, Complete mini

(Roche). Then the lysates were mixed with sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) sample buffer (Bio-Rad) with 5% 2-mercaptoethanol

(Wako) and boiled for 5 min. The samples were electrophoresed by SDS-PAGE on 5 to

20% gradient polyacrylamide gel, SuperSep Ace (Wako), and separated proteins were

blotted on a polyvinylidene difluoride membrane (Millipore). The membrane was

blocked for at least 1 h at room temperature with Tris-buffered saline containing 0.1%

38

Tween 20 (TBST) and 1% BSA. Then the membrane was incubated with a rabbit anti-

Src antibody (36D10: Cell Signaling) or mouse anti-Syk antibody (4D10.1: Abcam) in

TBST containing 1% BSA, followed by incubation with horseradish peroxidase-

conjugated donkey anti-rabbit IgG (H+L) or horseradish peroxidase-conjugated goat anti-

mouse IgG (H+L) (Jackson ImmunoResearch), respectively, and visualization by

Immobilon Western (Millipore). Band intensities were analyzed with a VersaDoc Imaging

System (Bio-Rad) and quantified with Image Lab version 3.0 software (Bio-Rad).

Phosphorylation assay. One μg/ml purified VLPs were treated with 20 μg/ml ZGP12/1.1

or CTR IgG for 1 h at room temperature. K562 cells were incubated with DMSO or 20

μM PP2 for 1 h at 37 ˚C. Untreated or PP2-treated K562 cells were inoculated with

untreated, CTR IgG-, or ZGP12/1.1-treated VLPs and incubated for 0, 10, 30, or 60 min

at 37 ˚C. At each time point, cells were collected and washed once in PBS and treated

with lysis buffer (0.1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 mM Tris HCl, pH

7.8) in the presence of a protease inhibitor cocktail, Complete mini (Roche), and a

phosphatase inhibitor cocktail, PhosSTOP (Roche). Then the lysates were mixed with

SDS-PAGE sample buffer (Bio-Rad) with 5% 2-mercaptoethanol (Wako) and boiled for

5 min. The samples were electrophoresed by SDS-PAGE on 5 to 20% gradient

polyacrylamide gel, SuperSep Ace (Wako), and separated proteins were blotted on a

polyvinylidene difluoride membrane (Millipore). The membrane was blocked for at least

1 h at room temperature with TBST and 1% BSA. Then the membrane was incubated

with a rabbit anti-phospho-Src family (Tyr416) antibody (Cell Signaling) in TBST

containing 1% BSA, followed by visualization using horseradish peroxidase-conjugated

donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch) and Immobilon Western

39

(Millipore). Band intensities were analyzed with a VersaDoc Imaging System (Bio-Rad)

and quantified with Image Lab version 3.0 software (Bio-Rad).

Statistical analysis. All data were analyzed using Excel software. In all experiments,

Student’s t-test was used to evaluate statistical differences. P values of less than 0.05 were

considered to be significant.

40

Results

Cytoplasmic tail of FcγRIIa is required for ADE of EBOV infection. To investigate

the role of FcγRIIa and, in particular, the importance of its cytoplasmic tail in EBOV

ADE, I compared the functions of wild-type FcγRIIa and FcγRIIaΔCT which is a deletion

mutant of FcγRIIa lacking its cytoplasmic tail. Both molecules were expressed on Jurkat

T cells, which are known to be poorly permissive for EBOV infection 80 and lack this Fc

receptor 81. Jurkat T cells were transduced with full-length FcγRIIa or FcγRIIaΔCT genes

using a retrovirus vector (Fig. 6A and B) and subsequently infected with VSV-EBOV GP

and infectious EBOV in the presence or absence of the GP-specific MAb ZGP12/1.1,

which is known to induce EBOV ADE 39 (Fig. 6C and D). I found that viral infectivity

was almost undetectable in naive and control vector-transduced Jurkat T cells but the

expression of wild-type FcγRIIa significantly enhanced the infectivity of VSV-EBOV GP

and EBOV in the presence of ZGP12/1.1, though not CTR IgG. Interestingly, the infection

rate of FcγRIIaΔCT-expressing cells was significantly lower than that of cells expressing

wild-type FcγRIIa. These results indicated that the FcγRIIa-MAb complex functioned as

a receptor-like molecule on this poorly permissive cell line and efficiently promoted

infection through the ADE of EBOV entry. More importantly, the results suggested that

signaling pathways via the FcγRIIa cytoplasmic tail were likely involved in the ADE of

EBOV entry into cells.

Cytoplasmic tail of FcγRIIa is important for enhanced viral uptake. FcγRIIa is

known to modulate phagocytosis/macropinocytosis through signaling pathways via its

cytoplasmic tail 82,83. Therefore, to analyze viral binding and intracellular uptake in more

detail, I produced DiI-labeled VLPs consisting of the major EBOV structural proteins,

41

Fig. 6. Importance of FcγRIIa and its cytoplasmic tail in EBOV ADE. (A) Schematic

representation of FcγRIIa and FcγRIIaΔCT. TM, transmembrane region; CT, cytoplasmic

tail. (B) Cell surface expression of the FcγRIIa ectodomain on Jurkat T cell lines. Jurkat

T cells stably expressing FcγRIIa and FcγRIIaΔCT were stained with an anti-CD32

antibody and analyzed by flow cytometry. Black lines represent vector-transduced,

FcγRIIa-, and FcγRIIaΔCT-expressing cells. Gray shading represents naive Jurkat T cells.

(C and D) Infectivity of VSV-EBOV GP and EBOV in Jurkat T cell lines. VSV-EBOV

42

GP (C) and EBOV (D) were incubated with medium alone (Control), CTR IgG, or

ZGP12/1.1, followed by inoculation into each Jurkat T cell line. At 24 h (C) and 72 h (D)

after inoculation, GFP-positive cells were counted. The relative percentage of infectivity

in each cell line was calculated by setting the IU value of untreated (Control), CTR IgG-,

and ZGP12/1.1-treated viruses in naive Jurkat T cells to 100%, respectively. The mean

and standard deviation of three independent experiments are shown. Statistical analysis

was performed using Student’s t-test (*p<0.05).

43

GP, VP40, and NP, and monitored the localization of VLPs in each transduced Jurkat T

cell line (Fig. 7). The number of VLPs attached to the surface of naive and empty vector-

transduced Jurkat T cells was not significantly different irrespective of the presence of

CTR IgG and ZGP12/1.1 (Fig. 7A and C). In contrast, the attachment of VLP was

significantly enhanced to similar extents in Jurkat T cells expressing FcγRIIa and

FcγRIIaΔCT in the presence of ZGP12/1.1 but not CTR IgG (Fig. 7A and C), suggesting

that the FcγRIIa ectodomain expressed on Jurkat T cells had the ability to increase the

VLP attachment mediated by ZGP12/1.1. Next, I assessed the number of VLPs

incorporated into intracellular vesicles along with Alexa Fluor 647-labeled dextran Mw

10,000 (Alexa647-labeled Dx10), a specific probe for visualizing phagocytotic and

macropinocytotic vesicles 11,84. After incubation for 2 h, ZGP12/1.1-treated VLPs

efficiently colocalized with Dx10 in Jurkat T cells expressing wild-type FcγRIIa, but not

in cells expressing FcγRIIaΔCT (Fig. 7B and D). Viral uptake was not observed

drastically in the absence of FcγRIIa and ZGP12/1.1. Furthermore, the number and size

of Dx10-positive vesicles incorporated into Jurkat T cells expressing FcγRIIaΔCT were

significantly smaller than those in cells expressing FcγRIIa, indicating the importance of

the FcγRIIa cytoplasmic tail in activating the phagocytosis/macropinocytosis (Fig. 7B

and E). These data suggested that the ADE infection of FcγRIIa-expressing Jurkat T cells

was associated with enhanced viral uptake into cellular vesicles, most likely due to the

activation of FcγRIIa-mediated signaling via its cytoplasmic tail.

Intracellular signaling via Src family PTKs contributes to ADE of EBOV infection.

To identify the intracellular signaling pathway involved in the ADE of EBOV entry, I

analyzed the effects of different inhibitors of signaling pathways in K562 cells, which

44

Fig. 7. Importance of FcγRIIa and its cytoplasmic tail in ADE-mediated VLP uptake

into cells. (A and B) Fluorescent images of attachment and internalization of VLP. (C-E)

45

Quantified fluorescent signals of VLP and Dx10. Untreated (Control), CTR IgG-, and

ZGP12/1.1-treated DiI-labeled VLPs were inoculated into each Jurkat T cell line and

incubated for 30 min on ice. After adsorption, the cells were incubated for 0 h (A and C)

or incubated with Alexa647-labeled Dx10 for 2 h at 37 ˚C (B, D, and E). VLPs (red) on

the cell surface (A and C) and VLPs (red) and Dx10 (green) in the cytoplasm (B, D, and

E) were monitored by confocal laser scanning microscopy. Scale bars represent 10 µm.

Nuclei of cells are visualized with DAPI (blue). The number of VLPs attached to the cell

surface (C), the colocalization of VLP (DiI) and Dx10 (Alexa647) signals (D), and the

number and size of Dx10 (E) were quantified. The number and size were calculated by

setting the value of FcγRIIa-expressing Jurkat T cells in the presence of ZGP12/1.1-

treated VLPs to 100%. The mean and standard deviation of three independent

experiments are shown. Statistical analysis was performed using Student’s t-test

(*p<0.05).

46

naturally express FcγRIIa. Consistent with previous studies 23,77, K562 cells were

permissive to VSV-EBOV GP and EBOV infections, and viral infection rates were

significantly enhanced in the presence of ZGP12/1.1 (Fig. 8). I then tested inhibitors of

Syk and Src family PTK (R788 and PP2, respectively) as these PTKs are known to be

principally involved in signaling pathways downstream of FcγRIIa, and in particular to

play important roles in inducing FcγR-mediated phagocytosis/micropinocytosis 74,85. I

found that the ADE of VSV-EBOV GP infection was significantly reduced in K562 cells

treated with these inhibitors in a dose-dependent manner. In contrast, only a limited

reduction was seen in non-ADE infection at the highest concentrations of the inhibitors

(Fig. 9). The ADE-specific inhibitory effect was more prominent in cells treated with PP2

than in those treated with R788.

Since Syk is reported to participate in the activation of signaling through PI3K,

Btk, and Ras, I further examined which pathways downstream of Syk contributed to the

ADE of VSV-EBOV GP infection using specific inhibitors of PI3K (LY294002), Btk

(LFM-A13), and Ras (Manumycin A) (Fig. 9). However, both ADE and non-ADE

infections by VSV-EBOV GP were dose-dependently reduced by LY294002 and

Manumycin A, respectively. LFM-A13 showed little effect on the infectivity of VSV-

EBOV GP. Subsequently, I confirmed the effects of these inhibitors using infectious

EBOV. Consistent with the data for VSV-EBOV GP, PP2 selectively reduced the ADE,

but not the non-ADE infection. Interestingly, R788 showed no effects on the ADE of

EBOV infection and rather enhanced the non-ADE infection. LFM-A13 slightly reduced

both the ADE and the non-ADE infections, whereas LY294002 and Manumycin A did

not inhibit the ADE infection, though Manumycin A slightly inhibited the non-ADE

infection.

47

Fig. 8. EBOV ADE in K562 cells. K562 cells were infected with VSV-EBOV GP or

EBOV following incubation with CTR IgG or ZGP12/1.1 for 30 min-1 h at 37 ˚C. After

incubation for 24 (VSV-EBOV GP) or 72 (EBOV) h, GFP-positive cells were counted

and IUs of viruses were determined. The relative percentage of infectivity was calculated

by setting the IU value of the viruses in CTR IgG-treated cells to 100%. The mean and

standard deviation of three independent experiments are shown.

48

Fig. 9. Identification of FcγRIIa-mediated signaling pathway for EBOV ADE. K562

cells were treated with the indicated concentrations of R788, PP2, LFM-A13, LY294002,

or Manumycin A for 1 h at 37 ˚C and inoculated with VSV-EBOV GP or EBOV

preincubated with CTR IgG, or ZGP12/1.1. The relative percentage of infectivity was

calculated by setting the IU value of the viruses in DMSO or ethanol-treated cells to 100%.

The mean and standard deviation of three independent experiments are shown. Statistical

analysis was performed using Student’s t-test (*p<0.05).

49

To further investigate the role of Src- and Syk-mediated signaling in EBOV ADE,

I generated Src and Syk knockdown K562 cells. K562 cells were transduced with

retroviral vectors expressing small hairpin RNAs (shRNAs) for silencing Src or Syk

genes (Fig. 10A and B) and infected with VSV-EBOV GP in the presence or absence of

ZGP12/1.1 (Fig. 10C). I found that transduced cells stably expressing Src shRNAs

(shSrc3 and shSrc4) showed approximately 50% reduction in protein levels (Fig. 10A)

and ZGP12/1.1-mediated ADE was significantly decreased in these cell lines (Fig. 10C).

In contrast, no significant difference was seen between ADE (ZGP12/1.1) and non-ADE

(CTR IgG) infections in Syk knockdown cells although 2 of the Syk shRNAs (shSyk3

and shSyk4) significantly reduced the expression of the Syk protein (Fig. 10B and C).

These results demonstrated that FcγRIIa-mediated signaling through the

activation of Src family PTKs contributed to the ADE of EBOV infection, but Syk-related

signaling including PI3K, Btk, and Ras did not seem to be specifically involved. I further

tested the effect of PP2 in K562/DC-SIGN or K562/hMGL, both of which have been

shown to act as attachment receptors for EBOV 77,86, and found that PP2 had limited

effects on the infectivity of VSV-EBOV GP in these cell lines (Fig. 11).

VLP-MAb complexes activate phosphorylation of Src in K562 cells. To directly detect

the activation of Src family PTKs, I quantified the phosphorylation levels of Src in K562

cells (Fig. 12). I found no significant difference in the cells inoculated with intact VLPs

alone at each time point. Likewise, inoculation of CTR IgG-treated VLPs did not enhance

Src phosphorylation levels. However, a significant increase of the Src phosphorylation

was detected at 30 and 60 min after K562 cells were exposed to ZGP12/1.1-treated VLPs

(Fig. 12 left). Furthermore, the enhanced phosphorylation was completely blocked by the

50

Fig. 10. Effects of Src and Syk knockdown on EBOV ADE. (A and B) Validation of

knockdown efficiency of Src and Syk in K562 cell lines. (C) Relative infectivity of VSV-

EBOV GP in K562 cell lines. Src (A) and Syk (B) proteins in each cell line were detected

by Western blotting. The band intensity of Src and Syk was standardized with that of the

β-actin band for each cell line. Relative intensities of the Src and Syk bands were then

calculated by setting the intensity of naive K562 cells to 100%. These cell lines were

infected with VSV-EBOV GP preincubated with CTR IgG or ZGP12/1.1 (C). The relative

percentage of infectivity was calculated by setting the IU value of the viruses in naïve

K562 cells to 100%. The mean and standard deviation of three independent experiments

are shown. Statistical analysis was performed using Student’s t-test (*p<0.05).

51

Fig. 11. Effects of PP2 on the VSV-EBOV GP infectivity in K562 cell lines stably

expressing C-type lectin. K562/DC-SIGN, K562/hMGL, and mock control K562 cells

were treated with DMSO or PP2 for 1 h at 37 ˚C and infected with VSV-EBOV GP in the

presence of the inhibitor. After incubation for 24 h, GFP-positive cells were counted. The

relative percentage of infectivity was calculated by setting the IU value of the virus in

DMSO-treated cells to 100%. The mean and standard deviation of three independent

experiments are shown.

52

Fig. 12. Increased phosphorylation of Src in K562 cells exposed to the VLP-

ZGP12/1.1 complex. Purified VLPs were preincubated with medium alone (Control),

CTR IgG, or ZGP12/1.1 and inoculated into K562 cells pretreated with DMSO or PP2

for 1 h at 37 ˚C. At 0, 10, 30, and 60 min post-inoculation, phosphorylation of Src at

Tyr416 (p-Src) was detected by Western blotting. The band intensity of p-Src was

standardized with that of the β-actin band at each time point. Relative intensities of the p-

Src bands were then calculated by setting the intensity of control cells (0 h) to 100%. The

mean and standard deviation of three independent experiments are shown. Statistical

analysis was performed using Student’s t-test (*p<0.05).

53

Src family PTK inhibitor, PP2 (Fig. 12 right). These findings suggested that Src were

activated by the interaction of the VLP-ZGP12/1.1 complex with FcγRIIa.

The ADE of EBOV entry depends on phagocytosis and/or macropinocytosis

mediated by Src family PTKs. To further characterize the role of the Src family PTK-

dependent signaling in the ADE of EBOV infection, I analyzed the effect of PP2 on the

attachment and uptake of DiI-labeled VLPs using K562 cells. I first compared the number

of VLPs attached to the cell surface among untreated, CTR IgG-, and ZGP12/1.1-treated

VLPs. Since the overexpression of FcγRIIa in Jurkat T cells increased the attachment of

VLPs to the cell surface in the presence of ZGP12/1.1 (Fig. 7), I hypothesized that

ZGP12/1.1 would enhance the VLP attachment to K562 cells. However, the number of

VLPs attached to the cell surface was not significantly different in the presence or absence

of ZGP12/1.1 (Fig. 13A left and D), and was not affected by the PP2 treatment (Fig. 13A

right and D), indicating that EBOV ADE in K562 cells did not result from increased viral

attachment to the cell surface. For the visualization of the VLP uptake into endosomes,

K562 cells expressing eGFP-Rab7, a late endosome marker, were used to analyze

colocalization of eGFP-Rab7 and internalized VLPs. I found that ZGP12/1.1-treated

VLPs were efficiently colocalized with eGFP-Rab7 in K562 cells, whereas only 10-20%

colocalization was seen in the cells inoculated with untreated or CTR IgG-treated VLPs

(Fig. 13B and E). I further found that the enhanced colocalization of eGFP-Rab7 and

ZGP12/1.1-treated VLPs was clearly blocked by the PP2 treatment (Fig. 13C and E).

These results indicated that the ADE of EBOV entry into K562 cells was dependent on

the Src family PTK activation leading to increased uptake of viral particles into cells.

Finally, I investigated whether the ADE of EBOV entry into K562 cells

54

depended on phagocytosis/macropinocytosis, which have been shown to be major

pathways of the EBOV entry through non-ADE pathways 11-13. I used Dx10 to visualize

phagocytotic and macropinocytotic vesicles in K562 cells and analyzed its colocalization

with VLPs. I found that approximately 70% of the ZGP12/1.1-treated VLP signals were

overlapped with Dx10 in intracellular vesicles (Fig. 14A and C). Furthermore, the

colocalization of Dx10 and ZGP12/1.1-treated VLPs was significantly blocked by the

PP2 treatment (Fig. 14B and C). To confirm that these observations were EBOV-specific,

I further analyzed DiI-labeled SUDV VLPs and found that ZGP12/1.1 affected neither

attachment/uptake of SUDV VLPs nor Dx10 uptake. (Fig. 15). These results suggested

that the surface-bound VLP-ZGP12/1.1 complex was incorporated into cells through Src

family PTK-dependent phagocytosis and/or macropinocytosis during the ADE of EBOV

entry.

55

56

57

Fig. 13. Enhanced VLP uptake into endosomes in K562 cells during ADE. (A-C)

Fluorescent images of attachment and internalization of VLP. (D and E) Quantified

fluorescent signals of VLP and Rab7. K562 cells expressing eGFP-Rab7 were incubated

with DMSO (A and B) or PP2 (A and C) for 1 h at 37 ˚C. Untreated (Control), CTR IgG-,

and ZGP12/1.1-treated DiI-labeled VLPs were inoculated into cells and VLPs (red) on

the cell surface at 0 h (A and D) and VLPs (red) and eGFP-Rab7 (green) in the cytoplasm

58

at 2 h (B, C and E) after adsorption were monitored by confocal laser scanning microscopy.

Scale bars represent 10 µm. Nuclei of cells are visualized with DAPI (blue). The number

of VLPs on the cell surface (D) and the colocalization of VLPs (DiI) and eGFP-Rab7

signals (E) were quantified. The mean and standard deviation of three independent

experiments are shown. Statistical analysis was performed using Student’s t-test

(*p<0.05).

59

60

Fig. 14. Colocalization of Dx10 and ZGP12/1.1-treated VLPs. (A and B) Fluorescent

images of internalization of VLP and Dx10. (C) Quantified fluorescent signals of VLP

and Dx10. K562 cells were incubated with DMSO (A) or PP2 (B) for 1 h at 37 ˚C.

Untreated (Control), CTR IgG-, and ZGP12/1.1-treated DiI-labeled VLPs were

inoculated into cells and incubated for 30 min on ice. After adsorption, cells were

incubated with Alexa647-labeld Dx10 for 1 h at 37 ˚C in the presence of DMSO (A) or

61

PP2 (B). VLPs (red) and Dx10 (green) in the cytoplasm were monitored by confocal laser

scanning microscopy. The colocalization of VLP (DiI) and Dx10 (Alexa647) signals was

quantified (C). Scale bars represent 10 µm. Nuclei of cells are visualized with DAPI

(blue). The mean and standard deviation of three independent experiments are shown.

Statistical analysis was performed using Student’s t-test (*p<0.05).

62

Fig. 15. Attachment, uptake, and localization of DiI-labeled SUDV VLPs. Untreated

(Control), CTR IgG-, and ZGP12/1.1-treated DiI-labeled SUDV VLPs were inoculated

into K562 cell lines and SUDV VLPs (red) on the cell surface at 0 h (A and D) and VLPs

(red) and eGFP-Rab7 (B and E) (green) or Dx10 (C and F) (green) in the cytoplasm at 2

h after adsorption were monitored by confocal laser scanning microscopy. Scale bars

63

represent 10 µm. Nuclei of cells are visualized with DAPI (blue). The number of SUDV

VLPs on the cell surface (D) and the colocalization of SUDV VLPs (DiI) and eGFP-Rab7

(E) or Dx10 (F) signals were quantified. The mean and standard deviation of three

independent experiments are shown. Statistical analysis was performed using Student’s

t-test (*p<0.05).

64

Discussion

It is well established that after the attachment of virus particles to cell surface

receptors a variety of signaling pathways are activated through tyrosine and

phosphoinositol kinases, and the subsequent cellular events such as endocytosis,

including macropinocytosis and phagocytosis, are important for the entry of viruses 87,88.

Likewise, it has been suggested that signaling pathways via FcγR are involved in ADE of

virus infections 68. However, there is limited information on the detailed molecular

mechanisms of intracellular signaling pathways required for ADE of virus infection. It

has been shown that the non-ADE entry of EBOV requires host factors such as PI3K, the

Rho family, and protein kinase C 11,12,89, although the virus-specific receptor molecules

involved in these signaling pathways are not yet identified. In the present study, I focused

on the ADE of EBOV entry and found that the FcγRIIa-mediated signaling pathway was

essential for this process, which is distinct from those required for the non-ADE entry.

The FcγRIIa cytoplasmic tail has been shown to be essential for ADE of dengue

virus entry and the involvement of Syk cascade in ADE entry has been reported 90-92.

These data indicate that the cytoplasmic tail of FcγRIIa is crucial for the ADE of EBOV

infection and that Src family PTK-dependent signaling is important to enhance viral

uptake into cellular vesicles during ADE-mediated entry of EBOV. Src family PTKs are

non-receptor tyrosine kinases involved in the regulation of diverse cellular functions like

proliferation, differentiation, adhesion, and phagocytosis 93,94. Importantly, this signaling

pathway is known to regulate endocytic machinery by triggering the reorganization of the

actin cytoskeleton and membrane remodeling 69,74. Indeed, previous studies have

demonstrated that Src family PTK-dependent signaling is required for the non-ADE entry

of some viruses into host cells 95,96. These data indicate that this signaling is also used to

65

promote viral particle uptake during the FcγRIIa-mediated ADE of EBOV entry.

It was noted that there was a significant increase (approximately 400%) in

infectivity of VSV-EBOV GP when FcγRIIaΔCT is expressed on Jurkat T cells, although

it was not as high as wildtype FcγRIIa. This observation suggest that the binding provided

by the external portion of FcγRIIa may also have some importance and that the signaling

function provided by the cytoplasmic portion of FcγRIIa further enhances the ADE effect.

It might also be possible that the FcγRIIa associated with lipid rafts activates some

FcγRIIa-mediated signals through its transmembrane domain as described previously 97.

Interestingly, I found that the Syk inhibitor R788 reduced the ADE efficiency of

VSV-EBOV GP but not EBOV. While the VSV pseudotype system is widely used to study

EBOV GP functions, it has been suggested that pseudotyped VSV and authentic EBOV

can utilize different entry pathways since the particle size and structure of pseudotyped

VSV do not accurately recapitulate those of EBOV 11,98. Thus, it may be possible that the

EBOV entry primarily relies on macropinocytosis as shown previously 11-13, whereas

VSV-EBOV GP can also be incorporated into smaller vesicles. I assume that this

difference could influence the effect of the inhibitor since Syk-dependent signaling might

be associated with caveolin-mediated endocytosis 99,100. Another difference found

between VSV-EBOV GP and authentic EBOV was that while the Syk inhibitor did not

affect EBOV ADE, non-ADE infection was significantly enhanced in the presence of this

inhibitor, an effect not observed for VSV-EBOV GP. This might be due to the effect on

post-entry mechanisms such as antiviral cellular responses, as proposed by a recent study

demonstrating that dengue virus-antibody complexes decreased type-I interferon-

stimulated gene expression triggered by the FcγR-mediated signaling pathway through

Syk, leading to enhanced replication of the virus 91. Since such an antiviral response could

66

be different between VSV- and EBOV-infected cells, it is possible that the Syk inhibitor

specifically affected the replication of EBOV, but not VSV, RNA genomes.

Previous studies have demonstrated that cellular C-type lectins such as DC-

SIGN and hMGL serve as attachment receptors and promote the entry of EBOV into cells

10,77,86. These C-type lectins, as well as Fc receptors, are thought to initiate phagocytic

pathways for uptake of microorganisms, cell debris, and apoptotic cells 101,102.

Interestingly, both C-type lectins and ADE-antibodies, including ZGP12/1.1, mainly bind

to the mucin-like region of EBOV GP, which has a number of N- and O-linked

glycosylation sites in the middle portion of the protein 23,77,86, suggesting a similarity in

the mechanism of the virus entry mediated by C-type lectins and ADE. However, the Src

family PTK inhibitor PP2 showed limited effects on the infectivity of VSV-EBOV GP in

the cell lines expressing DC-SIGN or hMGL. Taken together, my data suggest that the

FcγRIIa-mediated EBOV ADE principally depends on signaling pathways distinct from

those for C-type lectin-mediated entry, while both C-type lectins and ADE-antibody-

FcγRIIa complexes are assumed to serve as attachment receptors and subsequent

processes for membrane fusion (i.e., cathepsin cleavage and NPC1 binding) appear also

to be similar.

In conclusion, these data indicate that EBOV ADE is not simply dependent on

increased viral attachment through interaction between FcγRIIa and virus-antibody

complexes, and that the induction of FcγRIIa-mediated signaling associated with the

activation of Src family PTKs is essential for EBOV ADE. This signaling pathway most

likely promotes macropinocytosis, the major entry pathway of EBOV, and leads to

enhanced viral uptake into cells. The contribution of Src family PTKs to FcγRIIa-

mediated ADE of virus entry has not been demonstrated previously. Discovery of this

67

ADE mechanism provides a novel perspective for the general understanding of ADE of

virus infection. Although the impact of ADE on disease progression remains unclear for

many viruses, my findings may offer a potential new target to develop treatments for

ADE-associated diseases such as dengue hemorrhagic fever and possibly Zika virus

infection 38,103-105, since signaling pathways are known to be essential for virus entry into

cells and some signaling inhibitors have been considered to be potential treatment options

for virus infections 106-108. However, since non-ADE entry mechanisms of these viruses

are different from EBOV, it is required to investigate whether the Src family PTK-

mediated ADE mechanism can be generally applied for other viruses known to utilize

ADE entry into cells.

68

Summary

ADE of EBOV infection has been demonstrated in vitro, raising concerns about

the detrimental potential of some anti-EBOV antibodies. ADE has been described for

many viruses and mostly depends on the cross-linking of virus-antibody complexes to

cell surface Fc receptors, leading to enhanced infection. However, little is known about

the molecular mechanisms underlying this phenomenon. Here I show that FcγRIIa-

mediated intracellular signaling through Src family PTKs is required for ADE of EBOV

infection. I found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due

to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not

non-ADE infection, was significantly reduced by inhibition of the Src family PTK

pathway, which was also found to be important to promote

phagocytosis/macropinocytosis for viral uptake into endosomes. I further confirmed a

significant increase of the Src phosphorylation mediated by ADE. These data suggest that

antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling

pathway that leads to enhanced viral entry into cells, providing a novel perspective for

the general understanding of ADE of virus infection.

69

Conclusion

GP-specific neutralizing antibodies are known to play an important role in

protection against EBOV infection. However, the existing protective MAbs are ebolavirus

species-specific. Furthermore, some GP-specific antibodies increase EBOV infection in

vitro; indeed, ADE is considered a potential problem for the development of vaccines and

antibody therapy for EVD.

Thus, in chapter I, I developed and described MAb 6D6 which reduced

infectivities of representative isolates of all known ebolavirus species in vitro. Antigenic

variants escaping from 6D6 had amino acid substitutions within the IFL region which is

highly conserved in GPs of all ebolavirus species. MAb 6D6 inhibited the VLP-mediated

membrane fusion in endosomes, but did not affect the attachment and internalization of

VLPs. Furthermore, passive immunization with 6D6 showed protective effects against

EBOV and SUDV in mouse models. The discovery of this cross-neutralizing antibody

provides a promising option for broad-acting ebolavirus antibody therapy.

In chapter II, I demonstrated that FcγRIIa-mediated intracellular signaling is a

key factor for ADE of EBOV infection. The antibody-virus complex bound to the cell

surface FcγRIIa triggered the phosphorylation of Src family PTK that activated signaling

pathways leading to enhanced viral uptake into cells through phagocytosis and/or

macropinocytosis. These findings provide new insights into the mechanism of ADE-

mediated viral infections.

The present study provides important information useful for the design and

implementation of improved therapeutics and vaccines whose antiviral effects depend on

neutralizing antibodies. Furthermore, since for some viruses ADE has become a great

concern to disease control by vaccination and passive immunization, this study

70

demonstrates the novel perspective for the general understanding of ADE and it may also

offer a potential new cellular target to develop treatments for ADE-associated diseases.

71

Acknowledgments

I express my deepest gratitude to my supervisor, Prof. Ayato Takada (Division

of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido Univ.,

Sapporo, Japan), whose comments and suggestions were innumerably valuable

throughout the course of my study. He also provided many opportunities for me to

conduct field work abroad and participate in conferences.

I sincerely appreciate the invaluable support and advice provided by Prof.

Hirofumi Sawa (Division of Molecular Pathobiology, Research Center for Zoonosis

Control, Hokkaido Univ.), Prof. Hideaki Higashi (Division of Infection and Immunity,

Research Center for Zoonosis Control, Hokkaido Univ.), and Associate Prof. Kentaro

Yoshii (Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido

Univ.) during my research work.

I am deeply grateful to Associate Prof. Manabu Igarashi, Specially Appointed

Assistant Prof. Reiko Yoshida, and Specially Appointed Assistant Prof. Rashid Manzoor

(Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido

Univ.) for their technical and intellectual support.

I particularly thank Dr. Andrea Marzi, Dr. Heinz Feldmann, Ms. Key Menk, Dr.

Atsushi Okumura, Dr. Hideki Ebihara, Dr. Satoko Yamaoka (National Institute of Allergy

and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories), Dr.

Yoshimi Tsuda (Department of Microbiology, Graduate School of Medicine, Hokkaido

Univ.), and all the employees at Rocky Mountain Laboratories for their thoughtful advice

and cooperation. Working with them proved to be a truly valuable experience.

I am grateful to the coordinator of the Program for Leading Graduate Schools,

Hokkaido Univ., Prof. Motohiro Horiuchi (Laboratory of Veterinary Hygiene, Graduate

72

School of Veterinary Medicine, Hokkaido Univ.), and the members of the Leading

Program Office for their help with my PhD coursework.

I thank Dr. Masahiro Kajihara, Dr. Mieko Muramatsu, Dr. Osamu Noyori, Dr.

Makoto Kuroda, Dr. Junki Maruyama, Dr. Naganori Nao, and Ms. Hiroko Miyamoto

(Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido

Univ.) for their advice and suggestions. I also thank Mr. Nao Eguchi, Ms. Asako Shigeno,

Mr. Tatsunari Kondoh, Mr. Masahiro Sato, and Mr. Yoshihiro Takadate (Division of

Global Epidemiology, Research Center for Zoonosis Control, Hokkaido Univ.) for their

daily supports.

I wish to specially thank my friends, Ms. Minori Kuroda, Ms. Chiho Kaneko,

Ms. Reiko Akamatsu, Ms. Memi Muto, and Ms. Kanae Shiokawa, for their

encouragement and time. Finally, I express my gratitude to my family and my husband

for their understanding and wonderful support.

My research was funded by the Research Fellowship for Young Scientists from

the Japan Society for the Promotion of Science.

73

和文要旨

エボラウイルスは、フィロウイルス科に属し、ヒトを含む霊長動物に重

篤な出血熱を引き起こす病原体である。フィロウイルス科には、進化系統学的に

エボラウイルス属、マールブルグウイルス属、およびキュエヴァウイルス属に分

類される。マールブルグウイルス属およびキュエヴァウイルス属にはそれぞれ 1

種のみが知られているのに対し、エボラウイルス属は 5種(Zaire ebolavirus、Sudan

ebolavirus、Taï Forest ebolavirus、Bundibugyo ebolavirusおよび Reston ebolavirus)

に分けられている。2000 年以降、Zaire ebolavirus、Sudan ebolavirus および

Bundibugyo ebolavirus によるエボラウイルス病(EVD)がアフリカで頻繁に発生し

ている。これらのウイルスは抗原的に大きく異なっており、属間あるいは種間で

交差反応する免疫応答の誘導が困難なため、エボラウイルスに対する予防・治

療・診断法の開発にとって問題となっている。2014 年の西アフリカでの Zaire

ebolavirusによる EVD流行の際には、未承認薬ながら抗体療法が実施されたが、

使用されたモノクローナル抗体は Zaire ebolavirusによる EVDに対してのみ有効

である。

第 1章では、まず、Zaire ebolavirusおよび Sudan ebolavirusのウイルス様

粒子(VLP)で免疫した BALB/c マウスの脾臓を用いて、5 種のエボラウイルス全

てを中和する抗体の作出を試みた。フィロウイルスの表面糖蛋白(GP)を纏った

非増殖性シュードタイプ水胞性口炎ウイルス(VSV)に対する中和活性を指標に、

抗体産生ハイブリドーマ培養上清をスクリーニングした結果、全てのエボラウ

イルス種に対して高い中和活性を示すモノクローナル抗体(6D6)が得られた。次

に、6D6 存在下で、Zaire ebolavirus の GP 遺伝子をもつ増殖性シュードタイプ

VSVを培養することによってエスケープミュータントを選択し、GPのアミノ酸

配列を親株と比較したところ、エスケープミュータントの GPには、全てのエボ

74

ラウイルス種において高度に保存されている Internal fusion loop(IFL)のアミノ酸

配列内に変異が見られたことから、この領域がエピトープであると推察された。

共焦点顕微鏡を用いて蛍光(DiI)ラベルした VLP の細胞への吸着・取込み・膜融

合を抗体存在下および非存在下で比較した結果、6D6 はウイルスの細胞侵入過

程における膜融合を阻害することが判明した。さらに、6D6を治療的に投与した

マウスは Zaire ebolavirus の致死的感染に対し 100%が生残した。また、Sudan

ebolavirusを感染させたマウスにおいても、6D6の投与による治療効果が認めら

れた。これらの結果によって 6D6 の投与が複数のエボラウイルスに対して効果

的であることが判明した。

一方で、エボラウイルスの GPに対して誘導される抗体の一部は、in vitro

でウイルスの感染を増強することが知られている(抗体依存性感染増強現象：

ADE)。エボラウイルス感染におけるこの現象は、GPに対する抗体が Fcγ受容体

IIa(FcγRIIa)あるいは C1q と C1q 受容体を介して細胞とウイルスとを架橋するこ

とによって引き起こされると考えられているが、ADE の詳細なメカニズムは明

らかとなっていない。第 2章では、FcγRIIaを介する ADEに着目し、FcγRIIa下

流のシグナルと ADEとの関連について検討した。まず、エボラウイルス非感受

性細胞(Jurkat 細胞)を用いて、FcγRIIaまたはその細胞内領域を欠失させた変異体

(FcγRIIaΔCT)を強制発現させた細胞を作出し、ADEによる感染効率を比較した。

その結果、FcγRIIa の細胞内領域が ADE に重要であることが判明した。次に、

FcγRIIaを恒常的に発現している K562細胞を用いて、FcγRIIa下流の各種シグナ

ル阻害剤の効果を調べ ADEに必要なシグナルの特定を試みたところ、Src family

PTK特異的阻害剤である PP2 が、細胞内の Src family PTKのリン酸化を阻害す

ることによって ADEを介した感染のみを顕著に阻害することが判明した。最後

に、共焦点顕微鏡を用いて DiIラベルした Zaire ebolavirusの VLP の細胞への吸

75

着・取込みを抗体存在下および非存在下で比較した結果、ADE 抗体存在下で、

VLP の細胞への吸着数に変化は見られなかったが、細胞内への取り込み効率は

上昇していた。さらに、その上昇は PP2によって阻害された。また、細胞内に取

り込まれた VLP はマクロピノサイトーシス/ファゴサイトーシスマーカーとエ

ンドソームに共局在していた。これらの結果によって、ADE 抗体は FcγRIIa 下

流の Src family PTKsを介したシグナル伝達経路を活性化し、マクロピノサイト

ーシス/ファゴサイトーシスを誘導することによって細胞へのウイルスの取り込

み効率を上昇させ、その結果、感染増強が引き起こされていることが明らかとな

った。

本研究では、既知の全てのエボラウイルス種に対して交差中和活性をし

めすモノクローナル抗体を世界に先駆けて作出した。さらに、そのエピトープを

解析するとともに中和メカニズムを明らかにし、汎用性の高い EVD治療用抗体

医薬の開発に繋がる重要な知見を提供した。また、ワクチンや抗体医薬開発にと

って問題となる ADEのメカニズムを解析し、Src family PTKsを介したシグナル

伝達経路が重要であることを見出した。これらの知見は、今後 EVDの予防・治

療法開発に大きく貢献することが期待される。

76

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